The long term objective of this proposal is to elucidate the checkpoint signaling mechanism associated with the function of Rad17 in response to various forms of genotoxic stress in mammalian cells. Although Rad17 is believed to participate in the early stage of DNA damage recognition, the mechanism through which Rad17 transmits checkpoint signals is unknown. Recently, we demonstrated that the hRad17 function is regulated by the checkpoint kinases, ATR and ATM. Overexpression of a mutated hRad17 that can not be phosphorylated by the kinases abrogated the G2/M checkpoint in a dominant-negative manner and sensitized human fibroblasts to IR, UV radiation, and HU. Furthermore, we have uncovered a physical association between Rad17 and a serine-threonine phosphatase, PP5. Interestingly, a reduction in PP5 expression was found to abolish the IR induced ATM kinase activation, suggesting that the Rad17/PP5 complex may play a critical role in the activation of ATM in response to DNA damage. To firmly establish a signaling role for Rad17 in the transmission of checkpoint signals, we will focus our effort on two Specific Aims. For Aim 1, we will test the hypothesis that Rad17 phosphorylation by the two kinases represents a critical step in the initiation of downstream checkpoint signaling events leading to cell cycle arrest and DNA repair. Using an inducible system, we will determine the functional relationship between Rad17 and Rad1 complex in the mediation of checkpoint signals. By generating Rad17 deficient cells, we will test the dependency of ATM activation on the presence of Rad17, as well as the effect of Rad17 deficiency on genomic stability. For Aim 2, we will test the hypothesis that PP5 plays a critical role in regulating the activation of ATM. We will use mutant forms of PP5 to block or interfere with the function of PP5, as well as two different approaches to reduce or eliminate the expression of PP5, to probe the functional consequence of specific interactions between these proteins in the signaling responses to genotoxic stress. Cell cycle checkpoint proteins play critical roles in maintaining genomic stability and integrity to prevent the development of cancer and hereditary diseases. Thus, the accomplishment of these proposed studies will provide further understanding of the molecular mechanisms by which checkpoint signals are transmitted and contribute to the development of novel therapeutics for the treatment of cancer and other human diseases.